Strengthening the Gut Barrier Helps Prevent Overactivated Immune Systems

1. Strengthening the Gut Barrier Helps Prevent Overactivated Immune Systems Eric Weaver, PhD Proliant Health and Biologicals Ankeny, IA

2. Proteins vs. Bioactive Proteins

3. Background • Antibodies/immunoglobulins: Discovered in the 1900’s, textbook science • Immunoglobulins and other plasma proteins and peptides are essentialproteins for immunity! • Immunoglobulin (antibodies), sIgA, is the body’s primary means of defending the body in the oral cavity and digestive tract • >90% of all immunoglobulin is antibodies directed against intestinal antigens • Supplementation of immunoglobulin has not been feasible. • Technology and cost have been important factors • Dose is critical to the effect. • New processing and sourcing technologies have now made oral supplementation feasible.

4. Plasma Proteins Improves Growthin Weaned Pigs • Consistent improvement in young pig growth when plasma proteins are fed post-weaning. 47 trial summary • ADG 34% • ADFI 23% • G/F 9%

5. Species Organism Results Author Year Pigs E. coli  fecal scores Borg, et al. 1999 Pigs salmonella  fecal scores Borg, et al. 1999 Pigs E. coli  ADG,  mortality Bosi, et al. 2002 Pigs E. coli  ADG,  secretory IgA Bosi, et al. 2003 Pigs E. coli  ADG IRTA 2003 Pigs E. coli  shedding Deprez, et al. 1999 Pigs E. coli  ADG,  mortality IRTA 2002 Pigs rotavirus  diarrhea Harrell, et al. 2000 Pigs PMWS  survival,  ADG EMBRAPA 2005 Calves E. coli  survival,  ADG,  scours Quigley & Drew 2000 Calves crypto  scours,  shedding Hunt, et al. 2002 Calves corona  recovery Arthington, et al. 2002 Calves E. coli  survival,  ADG,  scours Nollet, et al. 1999 Shrimp WSSV  survival,  ADG Yukinori 2001 Trout Y. ruckeri  survival,  ADG Aljaro et al. 1999 Turkeys Pasteurella  survival,  ADG Campbell, et al. 2001 Summary of Plasma Protein Response in Challenge Trials

6. The Effect of Plasma Proteins on the Growth of Pair Fed Weaned Pigs +22%* Jiang et al., 2001

7. The Effect of Plasma Proteins on the Growth of Pair Fed Weaned Pigs +31%* +17%* +10%* Jiang et al., 2001

8. The Effect of Plasma Proteins on the Growth of Pair Fed Weaned Pigs * Jiang et al., 2001

9. The Effect of Plasma Proteins on the Growth of Pair Fed Weaned Pigs * * * Jiang et al., 2001

10. The Interaction Between TNF-α and IGF-I on Protein Synthesis

11. What is ImmunoLin®? The First Purified Immunoglobulin for Use in Supplements! • ImmunoLin® is a natural bovine immunoglobulin product isolated from edible bovine plasma. • Products: • ImmunoLin powder (>45% IgG) • ImmunoLin WP (>45% IgG) – agglomerated for use in liquid • Muscle Factors (>20% IgG) – edible-grade serum concentrate • Features: • Edible-grade, concentrated for lower inclusion rate • Light color, odor – blends well with other protein sources • Low plate count (aerobic plate count < 10,000, typical 1,000) • Low endotoxin

12. Frequently Asked Questions and Concerns • Is immunoglobulin digested or broken down which makes it not efficacious? • How can humans benefit from another species source of immunoglobulin?

13. Studies of IgG Recovery After Oral Administration * Represents % of original dose recovered in stool or an vitro sample

14. The Effect of Digestive Processes on the Biological Activity of Immunoglobulin Administered Orally • The average IgG recovery following digestion or simulated digestion in 12 published studies is 25% • No studies have reported 100% loss of IgG • Digestibility: In vivo studies indicate low relative digestibility of plasma proteins (75%-80%) when compared to milk protein (>95%). • IgG F(ab’)2 fragments also retain biological activity (LeFranc-Millot et al., 1995) • Clinical studies of bovine Ig and the large number of animal studies evaluating the efficacy of plasma proteins proves that Ig is biologically active following oral ingestion

15. Antigen-binding Capacity of Natural Immunoglobulin These data indicate that antibodies against common pathogens are found in high concentrations in ImmunoLin!

16. Immunity, the GI tract and Wellness • ..Adults have about 400 sq. meters of surface area and 2 lbs of bacteria in the GI tract so it is no wonder that the GI tract is the site where many health problems begin. It can be considered the most important immunological organ, accounting for 70% of the body’s immune cells.

17. Effects of Serum Proteins on Gut Permeability in a Cryptosporidiosis model Hunt et al., 2002

18. The Cyclical Consequences of Compromised Immunity and Barrier Function Immune Competence Barrier Function

20. Stress Heat Physical Psychological Ischemic Trauma – burns, surgery Nutrition and Diet Malnutrition of the gut Nutrient status Vit. D, Zinc, antioxidants Immune status Immune activation and inflammation Immunocompromise Factors Affecting Gut Barrier Function • Exercise • Age • Young and old • Genetics • NOD2 (IBD) • Drugs • Anti-inflammatories • Antibiotics • Disease • Infectious • Bacterial, viral, protozoal • Non-infectious • Pancreatic function

21. 10 Intestinal effects (on specific functions) Introduction Gut associated lymphoid tissue (GALT) Luminal Antigens (Food, microbes, toxins, etc)

22. Model of Immune Activation and Changes in Gut Barrier Function Water secretion Luminal Antigens (food, toxins, etc) Changes in tight junctions and epithelial function vascular permeability PP activation inflammatory cells infiltration MLN activation Systemic Immune Activation

23. Plasma protein concentrates attenuate the effects of S. aureus enterotoxin B on intestinal barrier function and immune system activation in an experimental model of inflammation Moreto, M., L. Russell, J. Polo, A. Perez-Bosque, C. Garriga, J. Campbell, and J. Crenshaw J. Nutr. 2006. Vol. 196:2838

24. 2 MHC II TCR Induction of intestinal inflammation Introduction S. aureus enterotoxin B (SEB) SEB Cytokine secretion Intestinal inflammation T-lymphocyte

25. 3 Inflammation model Experimental design Design SEB (i.p.) Day 30 Day 33 • Intestinal effects: • Unspecific • neutrophil infiltration • vascular permeability • water content in feces • On specific functions • organizedintestinal immune system • barrier properties • nutrient absorption Day 21 S 24 h 48 h Control diet + SDAP / +IC ON DIET 13-14 days 0 days • General / systemic effects: • feedconsumption and growth curve • analysis of hematological variables • concentration of serum IgA and IgG • spleen lymphocyte populations

26. Mucosal compartment Serosal compartment 13 Intestinal effects (on specific functions) Results Dextran & HRP flux * ∮ ∮ (40 kDa) Control = no challenge, standard diet SEB = toxin i.p. SEB-SDAP = toxin i.p. + plasma proteins in diet SEB-IC = toxin i.p. + immunoglobulin conc.

27. 34 B C * φ A * φ * φ φ Unpublished experiments Results Mucosal cytokine expression of pro-inflammatory cytokines • Pro-inflammatory cytokines • IL-6 • IFN – γ • TNF- α

28. * φ 13 * φ * φ Effects on Cytokines Results TNF-α

29. * 13 φ * φ φ φ Effects on Cytokines Results IFN-γ * ∮ (40 kDa)

30. Phase IV Report Diffuse GALT Lamina propria lymphocytes (LPL) Intraepithelial lymphocytes (IEL) T lymphocytes activated T lymphocytes Th lymphocytes Ts/c lymphocytes Tγδlymphocytes NK cells SEB SEB-SDAP SEB-IC SEB SEB-SDAP SEB-IC

31. Phase IV Report Comparison of the effects on diffuse & organized GALT Lamina propria lymphocytes (LPL) - 24 h Peyer’s patches (PP) - 48 h T lymphocytes activated T lymphocytes Th lymphocytes Ts/c lymphocytes Tγδlymphocytes NK cells SEB SEB-SDAP SEB-IC SEB SEB-SDAP SEB-IC (Phase IV Report) (J. Nutr. 2004 134: 2667–2672)

32. Phase V Report Cytokines and inflammatory mediators IL-10 IFN-γ TNF-α IL-6 LTB4 Peyer’s patches Mucosa Serum IFN-γ TNF-α IL-6 LTB4 IL-10 SEB SEB-SDAP SEB-IC SEB SEB-SDAP SEB-IC SEB SEB-SDAP SEB-IC

33. 15 Effects of SEB, PP and IC Conclusion • Conclusions: • SEB is a potent stimulant of the acute inflammatory response (innate immunity). • I.P. administration of SEB alters intestinal immune and barrier functions without causing systemic effects • PP/IC reduces the effects of SEB on: • Mast cell activation • Lymphocyte activation in GALT • Luminal water content • Intestinal permeability to dextran and HRP • PP/IC prevents the effects of SEB on pro-inflammatory cytokine In rats challenged with SEB, PP and IC reduces over-stimulation of the immune system and improves barrier function Proliant Health and Biologicals

34. Papers & communications • Plasma protein supplements attenuate the activation of mucosal intestinal lymphocytes induced by S. aureus enterotoxin B in rats. 10th European Nutrition Conference, Paris, July 10-13, 2007. • Dietary plasma protein supplements prevent the release of mucosal proinflammatory mediators in a rat model of intestinal inflammation. 10th European Nutrition Conference, Paris, July 10-13, 2007. • Dietary plasma supplementation reduces S. aureus enterotoxin B-induced intestinal inflammation in weaned rats. 2008. J. Nutr. 138:533-537. • Oral porcine plasma proteins prevent the release of mucosal pro-inflammatory cytokines in rats challenged with S. aureus enterotoxin B. Digestive Disease Week, 2008.

35. Hypotheses for the mechanism of action Immune stimulation by microflora or luminal antigens Immunoglobulins Glycoproteins LPS/endotoxin binding proteins Iron-binding, antiviral, antibacterial peptides Bacterial and viral aggregation and exclusion Endotoxin neutralization Bacteriostatic and viral inhibition activity Effectiveness of barrier function Reducing bacterial adhesion to the epithelium Access of luminal contents to dendritic cells and macrophages Nutrition of the epithelium Resulting from decreased immune system activation Immune tolerance Biological activity of plasma supplements (Tregs, IL-10)

36. Hypotheses for the mechanism of action Endotoxin Intestinal microorganisms immune stimulation by microflora or endotoxin Intestinal lumen IEL effectiveness of barrier function Tissue destruction Naive T-cell APC Lamina propria Pro-inflammatory Activated T-cell immune tolerance Cell recruitment Granulocytes Regulatory T-cell Anti-inflammatory Th1 cells Pro-inflammatory Th2 cells Pro-inflammatory Macrophages Lymphocytes

37. Immune Compromise or Loss in Gut Barrier Function Microbial Translocation and Endotoxemia Immune Activation and Inflammation Appetite Catabolism Insulin/IGF-I Resistance* Weight Loss Lean Tissue *O’Connor et al. 2008. Regulation of IGF-I function by proinflammatory cytokines: At the interface of immunology and endocrinology

38. When can oral immunoproteins be beneficial? • Inflammatory events: new bacterial (e.g. traveler’s diarrhea) or viral exposure, and inflammatory disease, a stimulus for loss of barrier function and over response to LPS • Stress: Suppresses immune response/barrier function • Geriatrics: Inflammation increases with age, gut barrier function declines • Young: Immunonaive, deprived of colostrum or breast milk, premature weaning, ENC status • Exercise: Increases endotoxemia and inflammatory cytokine production, increases risk of heat stress/stroke, can adversely affect training and/or performance • Surgery/hospitalization – increased TNF-α lowers survival • Gut health issues: diarrhea, ulcers (Helicobacter), and NSAIDS increase gut permeability • Immune deficient – disease, weaning, gut malnutrition, steroid therapy, and antibiotic use can all compromise intestinal immunity *Ideal supplement forms: drink mixes, capsules, chewable tablets, syrups, and bars *References available upon request

39. Causes and Consequences of Heat Stress-Induced GI Barrier Dysfunction G.P. Lambert. Exerc. Sport Sci. Rev. 185-190, 2004.

40. Ideas for use in Supplements Consumers with need for greater immunity and barrier function 1) Travelers: New exposure (air, food and water), high stress. 2) Retailers: Constant exposure to the public. 3) Stressed: No time to be sick. Stress compromises immunity. 4) Elderly: Help for an immune system that doesn’t respond. Prefer liquids or bars. 5) Children: A boost when they need it. Limits on taste, dosage form and safety issues. 6) Athletes – improve health in training and competition. 7) Susceptible: Powerful support for the immune deficient. Delivery systems compatible with consumer and Ig: Capsule or tablet Individual serve packets (drink or consume as is) Cannister of 30 d supply Chewable tablets (500 mg per tablet), 10/roll Dose is important! Supplements: 2.5 - 10 grams per serving based on need! Functional foods: 250 mg to 1,500 mg per serving *Goals of supplements and functional foods are different. Functional foods have improved health characteristics vs traditional food products. The objective for supplements is to deliver a nutrient or bioactive ingredient at levels not normally in foods in sufficient quantities to improve health. *References available upon request

41. Who can benefit? Applications in Gastrointestinal Wellness 1) IBS/Stressed: Imbalance in the gut. “D” form affects half of this group. 2) AIDS/Immune deficient: Chronic problems with diarrhea. 3) Elderly: Help for an immune system that doesn’t respond. Combine with fiber? 4) Children: Viral GI infections are common. Combine with a immune nutrients such as zinc 5) Travelers: New exposure, high stress. 6) Recovery Hospitalized: Need to maintain lean body mass, add to whey-based beverage Ulcers - NSAIDS, aphthous Antibiotics Medications: chemotherapy *References available upon request

42. Ideas for use in Supplements Delivery systems compatible with consumer and Ig: Capsule or tablet Powder - individual serve packets (drink or consume as is) Cannister containing 30 d supply Chewable tablets (500 mg per tablet), 10/roll Formulation considerations: Immune support – nutrients compatible for age groups and situations Gut health – compatible with probiotics (feed and protect) Inflammation – all system approach that includes the gut Dose is important! Supplements: 2.5 - 10 grams of ImmunoLin per serving. Recommendation for adapting dose. Functional foods: 250 mg to 1,500 mg per serving *Goals of supplements and functional foods are different. Functional foods have improved health characteristics vs traditional food products. The objective for supplements is to deliver a nutrient or bioactive ingredient at levels not normally in foods in sufficient quantities to improve health. *References available upon request

43. Current issues supporting opportunities for ImmunoLin • Recent studies Gut health and inflammation – Perez-Bosque et al. J. Nutr. 2004, 2006, and 2007 HIV – lack of immunity in the gut, even with years of ART treatment – Mehandru et al. PLoS 2006; Brenchley et al., 2007 Chemotherapy damages intestinal barrier dysfunction – Song et al – Dig Dis Sci 2006 Plasma proteins assist in recovery from rotavirus-induced diarrhea – Corl et al. (2007) • Current issues support marketplace opportunities Food-borne disease, travel illness, HIV, IBD, chemo- and antibiotic-induced diarrhea continue to be major health problems world-wide • Support of Medical Advisory Board

44. Comparing ImmunoLin to other immune ingredients

45. Summary: Why supplementation works!! • Immunoglobulins are essential proteins for immunity! • ImmunoLin is an effective boost to sIgA, a natural constituent of the body • ImmunoLin provides antibodies that the body needs • Immunoglobulin survives digestion. • ImmunoLin is a powerful aid to preserving gut barrier function • ImmunoLin reduces the burden on the immune system • Nutrients in the body can be used for “productive” functions, not to fuel the immune response • The body’s immune system can better defend the body against “other” agents.